Ever since the Raman Effect was discovered, it has played a significant role in the field of vibrational spectroscopy. Although Raman scattering acts as an effective tool to probe molecular structure, it has some disadvantages like fluorescence overlap, small cross-sections and low sensitivity. Of late, due to the emergence of nanotechnology and improvement in sensitivity of optical instruments, Surface Enhanced Raman Scattering (SERS) has played an important role in overcoming the above-mentioned disadvantages. In this phenomenon, the Raman mode intensity of a molecule is enhanced by several orders of magnitude (˜106 to 1014) upon adsorption of the molecule to noble metal surfaces, which exhibit atomic scale roughness. Ever since its discovery, SERS has been utilized as an effective analytical tool to study molecules of biological interest and detection tool to probe different aspects of biology. Most importantly, it has been used to study protein-drug interaction and DNA complexes with a fair amount of success. SERS is used to unveil structural information of proteins, such as p300, which are not accessible to crystallographic techniques. SERS is equipped with a high degree of sensitivity to detect single molecules within a confined volume. One of the most important aspects of the SERS study is the experimental setup used to probe and detect the analyte under supervision. Although commercial Raman microscopes have played a key role in the success of SERS, most of them are expensive, sophisticated and lack flexibility. There have been a few successful attempts to build Raman microscopes previously, but most of them require specialized knowledge for construction. Therefore, it is important to design SERS set-up which is not only inexpensive, but also versatile to perform multiple experiments. Here in the instant invention setting up of SERS spectrometer using a simple viewing microscope with an epifluorescence attachment along with a mirror.
The fluorescence microscopes used in SERS are provided with dichroic mirror-cube holders. For fluorescence measurements a dichroic mirror is used, which is selected based on the excitation and emission band of the chromophore. In the case of Raman spectroscopy, this effectively blocks a large region of the Raman spectrum (˜200 cm−1) close to the Rayleigh scattering. There is an added disadvantage of using the dichroic mirror as it also cutoff the high-frequency Raman spectra. The instant invention of mirror overcomes the problem faced by dichroic mirrors, wherein the dichroic mirrors of the fluorescence microscope are replaced with a special mirror.